Microscope, focusing unit, fluid holding unit, and optical unit

ABSTRACT

A microscope includes: a sample placement part having a placement surface on which to place a sample and a bottom face opposite to the placement surface; an observation lens; and an optical unit for generating sheet light and use of the microscope. The microscope of an embodiment is arranged such that the sheet light enters the sample placement part from the bottom face and passing through the sample placement part to irradiate the sample, and fluorescence from the sample passes through the sample placement part toward the bottom face to be received by the observation lens. The microscope can, with this arrangement, utilize the advantages of a SPIM, and allows observation of a sample which observation is free from a restriction of the size of a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This U.S. non-provisional application is a continuation of U.S.application Ser. No. 15/854,884, filed on Dec. 27, 2017, which is acontinuation of U.S. application Ser. No. 14/781,625 filed on Oct. 1,2015, which is the U.S. National Phase application of PCT ApplicationNo. PCT/JP2014/059883 filed on Apr. 3, 2014, which claims priority toJapanese Application No. 2013-079956 filed on Apr. 5, 2013, the contentsof each of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a microscope, a focusing device for usein a microscope, a fluid holding device for use in a microscope, and anoptical unit for use in a microscope.

BACKGROUND ART

There has been known a microscope that irradiates an observation planeof a sample locally with light and that receives fluorescence radiatedfrom the sample. The microscope, with such an arrangement, allows anobservation of sectioned planes of a sample. Such a microscope, which iscapable of preventing light from illuminating that portion of a samplewhich is not an observation plane, can reduce the background during theobservation of the sample and can also reduce, for example, harmfulaction to a sample or attenuation of fluorescence that are caused byirradiation of the sample with excitation light.

Patent Literatures 1 and 2 and Non Patent Literature 1 each disclose ahighly inclined and laminated optical sheet microscopy for an opticalmicroscope, with which technique illumination light is refracted withuse of an objective lens to irradiate a sample in an oblique directionwith respect to the optical axis of the objective lens. FIG. 18 showsenlarged views of part of an optical microscope that uses the highlyinclined laminated optical sheet microscopy of Patent Literature 1. In acase where an optical microscope is arranged such that illuminationlight enters a sample in a direction so angled as to be close toperpendicular to the optical axis of the objective lens as illustratedin FIG. 18, the optical microscope can irradiate a sample with a thinlayer-shaped light having a small thickness along the direction of theoptical axis of the objective lens. According to Patent Literature 1,the optical microscope is capable of continuously capturing an image ofa sample while moving the focal position of the objective lens toproduce a three-dimensional image at a high resolution. PatentLiterature 1 discloses in particular that the optical microscope iscapable of imaging at a single-molecule level.

The technique disclosed in Patent Literature 1 may serve to adjust theposition on an objective lens at which position illumination lightenters the objective lens, so that the angle θ between optical axes ofillumination light refracted and the objective lens is close to 90degrees. The technique, however, does not allow the angle θ to beexactly 90 degrees. Thus, the optical axis of illumination light is notparallel to the observation plane of the objective lens. Thisarrangement results in an image being ununiformly out of focus tovarying degrees over the observation plane, the image thus havingdecreased quality evenness. Further, the above arrangement letsillumination light illuminate a region of a sample which region is otherthan the observation plane, and thus increases the background, with theresult of a fluorescence image having a decreased resolution. Inparticular, in a case of producing a three-dimensional image, such animage will have a low resolution along the direction parallel to theoptical axis of the objective lens.

Non Patent Literature 2 discloses SPIM (selective plane illuminationmicroscope), which irradiates a sample with a thin layer-shaped sheetlight, collects radiated fluorescence with use of an objective lenshaving an optical axis perpendicular to the optical axis of the sheetlight, and forms an image of the collected fluorescence with use of acamera. In a case of observing a sample with use of the SPIM of NonPatent Literature 2, the SPIM rotates agarose gel in which the sample isembedded and thus irradiates the sample with sheet light from variousangles, and captures an image of the sample with use of a camera. FIG.19 is a diagram schematically illustrating a part of the microscope ofNon Patent Literature 2 on which part a sample is placed. PatentLiterature 3 discloses that in a case of collecting light with use of anillumination lens and irradiating a sample with the collected light inthe form of layer-shaped sheet light, the sheet light has a thicknessalong the direction parallel to the optical axis of the objective lenswhich thickness depends on the numerical aperture of the illuminationlens. This disclosure indicates that in a SPIM, increasing the numericalaperture of the illumination lens can reduce the thickness of sheetlight and can thereby reduce the background.

Patent Literatures 4 to 6 each similarly disclose a microscope thatcollects fluorescence from a sample with use of an objective lens havingan optical axis perpendicular to the optical axis of sheet light andthat thereby produces a three-dimensional image of the sample.

In a case of irradiating a sample with sheet light in the directionperpendicular to the optical axis of an objective lens as with themicroscopes disclosed in Patent Literatures 3 to 6 and Non PatentLiterature 2, it is possible to reduce the background and, as a result,obtain a high resolution.

CITATION LIST

-   Patent Literature 1-   Japanese Patent Application Publication, Tokukai, No. 2003-185930 A    (Publication Date: Jul. 3, 2003)-   Patent Literature 2-   Japanese Patent Application Publication, Tokukai, No. 2005-3909 A    (Publication Date: Jan. 6, 2005)-   Patent Literature 3-   Japanese Translation of PCT International Application, Tokuhyo, No.    2006-509246 (Publication Date: Mar. 16, 2006)-   Patent Literature 4-   Japanese Patent Application Publication, Tokukai, No. 2012-93757 A    (Publication Date: May 17, 2012)-   Patent Literature 5-   Japanese Patent Application Publication, Tokukai, No. 2012-108491 A    (Publication Date: Jun. 7, 2012)-   Patent Literature 6-   U.S. Patent Application Publication No. US2011/0304723 (Publication    Date: Dec. 15, 2011)-   Non Patent Literature 1-   M. Tokunaga et al., Nature Methods, 5, pp. 159-161, 2008-   Non Patent Literature 2-   Jan Huisken et al., SCIENCE 2004 VOL 305, pp. 1007-1009, 2004

SUMMARY OF INVENTION Technical Problem

The existing SPIM allows observation of a sample (for example, acultured cell) embedded in agarose gel as with the SPIM of Non PatentLiterature 2 illustrated in FIG. 19. A sample such as a cell in aculture solution cultured in a petri dish is thus difficult to observewhile the sample is being cultured. This means that conventional SPIMshave only limited measurement applications. Further, since a culturedcell embedded in gel may behave differently from when the cell is in aculture medium, conventional SPIMs are not suitable for continuousobservation of a living cell. In addition, conventional SPIMs, whichrequire a sample to be embedded in a gel having a predetermined volume,are problematically limited in terms of the size of a sample.

In view of that, the inventors of the present invention have studied thepossibility of not embedding a sample in gel and observing the sampleplaced on a flat cover slip, similarly to a biological sample for normalmicroscope observation, with use of a SPIM configuration. In this case,a detection objective lens may be oriented perpendicularly to thesurface of the cover slip so that a fluorescence image will not beinfluenced by aberration that occurs when fluorescence passes throughthe cover slip.

Sheet light is, however, wide along the direction parallel to theoptical axis of the objective lens. Thus, in a case where lightirradiates a region near the cover slip, a portion of such light passesthrough the top and bottom faces and end faces of the cover slip.Individual light beams thus travel on optical paths different from oneanother due to refraction, reflection, and/or the like, so that thesheet illumination has decreased quality. Sheet light irradiating asample thus has an increased thickness, with the result of a failure tosufficiently utilize the advantages of a SPIM.

The present invention has been accomplished in view of the aboveproblem. It is an object of the present invention to provide amicroscope having high resolution which microscope can utilize theadvantages of a SPIM, allows observation of a sample which observationis free from a restriction of the size of a sample, and allowsobservation of, for example, a cell in a culture solution or a sample ona cover slip and a member included in the microscope.

Solution to Problem

In order to solve the above problem, a microscope of the presentinvention is a microscope, including: a sample placement part having aplacement surface on which to place a sample and a bottom face oppositeto the placement surface; an observation lens for receiving fluorescencefrom the sample; and an optical unit for generating sheet lighttraveling in a direction parallel to an observation plane of theobservation lens, the sheet light entering the sample placement partfrom the bottom face and passing through the sample placement part toirradiate the sample, the fluorescence passing through the sampleplacement part toward the bottom face to be received by the observationlens.

The above arrangement allows an observation to be carried out withoutthe need to embed a sample on the sample placement part in gel and in astate where the advantages of a SPIM are utilized. Further, since theabove arrangement allows the observation lens to be so disposed as to beopposite to a sample with respect to the sample placement part, the sizeof a sample placed on the sample placement part is not limited to a sizewithin the range of the working distance of the observation lens.

In a case where a SPIM observation is to be carried out with anobjective lens placed on an axis perpendicular to a surface of thesample placement part, sheet light will interfere with the sampleplacement part, with the result of a decrease in the quality of thesheet illumination. In contrast, the microscope of the presentinvention, which allows all the light to enter the observation planefrom the bottom face of the sample placement part, makes it possible tosuccessfully control the thickness of sheet light even in a case where asample near the sample placement part is to be observed.

The microscope of the present invention may further include: a focusingmechanism for adjusting a relative positional relationship between theobservation lens and the sample, wherein the focusing mechanism changesthe positional relationship along a first direction, which is parallelto an optical axis of the observation lens, and a second direction and athird direction, which define the placement surface.

With the above arrangement, adjusting the relative positionalrelationship between the observation lens and the sample placement partalong the second direction and third direction can change the observedfield of view without changing the distance between the sample placementpart and the observation lens. The above arrangement can also preventunnecessary contact between the sample placement part and theobservation lens while the microscope changes the observed field ofview. At the same time, adjusting the relative positional relationshipbetween the observation lens and the sample placement part along thefirst direction can achieve focusing without changing the observed fieldof view. The above arrangement thus makes it possible to capture imagesof a sample sectioned on a plurality of observation planes perpendicularto the first direction to produce a three-dimensional image of thesample at a high resolution.

The microscope of the present invention may further include: a fluidholding device for holding fluid between the observation lens and thesample placement part, wherein: the fluid holding device includes aconnection section for connection to the observation lens; an end faceso designed as to face the bottom face; a transmission window throughwhich the sheet light enters the sample placement part; and a fluidholding section for holding fluid inside itself; and in a state wherethe fluid holding device is connected to the observation lens, the endface is substantially parallel to the bottom face, the sheet lightpasses through the transmission window and then through the fluidholding section to irradiate the sample, and the fluorescence passesthrough the fluid holding section to be received by the observationlens.

For measurement at a higher resolution and higher sensitivity, themicroscope of the present invention may preferably be arranged such thatthe observation lens is an immersion lens; and the fluid holding sectionis filled with a liquid corresponding to the immersion lens.

In a case where an immersion lens is used as the observation lens, aliquid corresponding to the immersion lens should be present between thebottom face of the sample placement part and the lens. The microscope ofthe present invention is arranged such that the bottom face of thesample placement part is not perpendicular to the optical axis of theobservation lens. Thus, it may not be easy to hold the liquidcorresponding to the immersion lens between the bottom face and the lenswithout moving (or changing the shape of) the liquid when changing therelative positional relationship between the observation lens and thesample placement part. With the above arrangement, however, the fluidholding device can hold the liquid between the observation lens and thesample placement part in a state where the surface shape of the liquidis maintained.

The microscope of the present invention may preferably be arranged suchthat the fluid holding section is filled with water; and the sampleplacement part has a refractive index of 1.28 to 1.38.

The microscope is arranged such that the observation lens has an opticalaxis not orthogonal to the placement surface. In such a case, left-rightasymmetric aberration will emerge depending on the difference betweenthe refractive index of the liquid filling the fluid holding section andthe refractive index of the sample placement part. However, in a casewhere the fluid holding section is filled with water, and the sampleplacement part has a refractive index of 1.28 to 1.38, which isequivalent to the refractive index of water, it is possible to reduceaberration significantly and form a stable image.

The microscope of the present invention may preferably be arranged suchthat the observation plane and the placement surface form an anglewithin a range from 1 degree to 75 degrees.

The angle between the observation plane, which is parallel to thetraveling direction of sheet light, and the placement surface needs toallow generation of sheet light having a sufficient thinness. The anglebetween the observation plane and the placement surface is thuspreferably so adjusted as to fall within the range from 1 degree to 75degrees in correspondence with the purpose of measurement.

The microscope of the present invention may preferably be arranged suchthat the optical unit includes an optical surface plate having a surfaceon which one or more optical elements for generating the sheet light aredisposed; and the optical surface plate is oriented so that the surfaceof the optical surface plate is parallel to the observation plane.

With the above arrangement, the one or more optical elements aredisposed on the surface of the optical surface plate. This allows theoptical unit to generate sheet light traveling in a direction parallelto the observation plane.

The microscope of the present invention may preferably be arranged suchthat the one or more optical elements include a light source, a lightconverging element, and a light distributing element; and the one ormore optical elements optionally further include a wedge prism.

In order to solve the above problem, a focusing device of the presentinvention is a focusing device for, in a case where a sample on aplacement surface of a sample placement part is to be observed,adjusting a relative positional relationship between an observation lenshaving an optical axis not orthogonal to the placement surface and thesample, the focusing device including: a first stage for changing therelative positional relationship between the observation lens and thesample along a first direction, which is parallel to the optical axis ofthe observation lens; a second stage for changing the relativepositional relationship between the observation lens and the samplealong a second direction and a third direction, which define theplacement surface; and a jig for fixing the sample placement part, thejig being attached to one of the first stage and the second stage, theone of the first stage and the second stage, to which one the jig isattached, being placed on one of the first stage and the second stage towhich one the jig is not attached.

In order to solve the above problem, a fluid holding device of thepresent invention is a fluid holding device for holding fluid between anobservation lens and a sample placement part having a placement surfaceon which to place a sample to be observed and a bottom face so designedto be opposite to the placement surface, the fluid holding deviceincluding a connection section for connection to the observation lens;an end face so designed as to face the bottom face; a transmissionwindow through which sheet light enters the sample placement part; afluid holding section for holding fluid inside itself, in a state wherethe fluid holding device is connected to the observation lens, the endface being substantially parallel to the bottom face, the sheet lightfrom an optical unit passing through the transmission window and thenthrough the fluid holding section to irradiate the sample, andfluorescence from the sample passing through the fluid holding sectionto be received by the observation lens.

In order to solve the above problem, an optical unit of the presentinvention is an optical unit including an optical surface plate having asurface on which one or more optical elements for generating sheet lightare placed, the optical surface plate being oriented so that the surfaceof the optical surface plate is parallel to an observation plane of anobservation lens.

Advantageous Effects of Invention

With one mode of the present invention, it is possible to observe asample on the sample placement part without embedding the sample in geland in a state where the advantages of a SPIM are utilized. Further, thesize of a sample placed on the sample placement part is not limited to asize within the range of the working distance of the observation lens.This makes it possible to observe a sample on a transparent planarsubstrate (sample placement part) similarly to a normal biologicalmicroscope, and thus allows observation of, for example, a relativelylarge biological sample such as an individual mouse.

The above arrangement further prevents a decrease in the quality ofsheet light even in a case where an illuminated region is formed nearthe placement surface of the sample placement part. Thus, even in a casewhere, for example, a sample adherent to the sample placement part is tobe observed, it is possible to observe the sample in a state where theadvantages of a SPIM are utilized. For instance, it is possible todirectly observe an adherent cell cultured in a petri dish.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram schematically illustrating a main configuration of amicroscope of one embodiment of the present invention.

FIG. 2 is a diagram schematically illustrating a main configuration of amicroscope of one embodiment of the present invention.

FIG. 3 is a diagram schematically illustrating an overall configurationand an optical path of a microscope of one embodiment of the presentinvention.

FIG. 4 illustrates shapes of sheet light of a microscope of oneembodiment of the present invention.

FIG. 5 illustrates images of a sample that are produced with use of amicroscope of one embodiment of the present invention and fluorescenceintensity distributions of the sample.

FIG. 6 shows diagrams each schematically illustrating a configuration ofa microscope of a comparative example.

FIG. 7 is a diagram schematically illustrating a configuration of amicroscope of one embodiment of the present invention.

FIG. 8 shows diagrams each illustrating how an image changes in responseto a change in the positional relationship between an observation lensand sample placement part of a microscope of one embodiment of thepresent invention.

FIG. 9 shows diagrams each schematically illustrating a mainconfiguration of a microscope of one embodiment of the presentinvention.

FIG. 10 shows diagrams each schematically illustrating a mainconfiguration of a microscope of one embodiment of the presentinvention.

FIG. 11 shows diagrams illustrating aberration correction by amicroscope of one embodiment of the present invention.

FIG. 12 shows diagrams each illustrating an optical path and a luminancedistribution of a light beam in an optical unit.

FIG. 13 is a diagram schematically illustrating an overall configurationand an optical path of a microscope of a variation of the presentinvention.

FIG. 14 shows diagrams each illustrating an arrangement for bending alight beam in an optical unit.

FIG. 15 shows diagrams each illustrating optical paths of light beamshaving different wavelengths which light beams are bended with use of awedge prism in an optical unit.

FIG. 16 illustrates an Example involving use of a microscope of thepresent invention.

FIG. 17 illustrates an Example involving use of a microscope of thepresent invention.

FIG. 18 shows diagrams each schematically illustrating a mainconfiguration of an optical microscope based on a conventional highlyinclined laminated optical sheet microscopy.

FIG. 19 is a diagram schematically illustrating a main configuration ofa conventional SPIM.

DESCRIPTION OF EMBODIMENTS Embodiment 1

The description below deals in detail with an embodiment of the presentinvention with reference to FIGS. 1 through 5.

FIG. 1 is a diagram schematically illustrating a main configuration andan optical path of a microscope 1 of the present embodiment. Themicroscope 1 of the present embodiment, as illustrated in FIG. 1,includes a sample placement part 10 for placing a sample, an opticalunit 20 for irradiating the sample with light, and an observation lens31 for receiving light radiated from the sample. FIG. 1 illustrates anillumination lens 26 as a member of the optical unit 20 whichillumination lens 26 is disposed at an opening for emission of lightfrom the optical unit 20.

The sample placement part 10 has a placement surface 11 on which toplace a sample 12 and a bottom face 13 opposite to the placement surface11. The microscope 1 of the present embodiment is, as illustrated inFIG. 1, so configured as to be usable as an inverted microscope suchthat the illumination lens 26 and the observation lens 31 are sodisposed as to be opposite to the sample 12 with respect to the sampleplacement part 10 (that is, with respect to the plane including theplacement surface 11). The sample placement part 10 is entirely orpartly made of a light-transmitting material. The sample placement part10 may be a single cover slip or a bottom face of a culture plate(including a multiwell plate and a microfluidic device) or part of thebottom face. The optical unit 20 irradiates a sample 12 with sheet lightthrough the bottom face 13 of the sample placement part 10. The opticalunit 20 emits sheet light, which enters the sample placement part 10 atthe bottom face 13 and then exits the sample placement part 10 at theplacement surface 11, to finally irradiate the sample 12. Theobservation lens 31 receives fluorescence radiated from the sample 12through the bottom face 13 of the sample placement part 10. The sample12 radiates fluorescence, which enters the sample placement part 10 atthe placement surface 11 and then exits the sample placement part 10 atthe bottom face 13, to finally be received by the observation lens 31.Further, the illumination lens 26 and the observation lens 31 are sooriented as to have optical axes that are orthogonal to each other on aplane (that is, the X-Z plane in FIG. 1). This arrangement allows theoptical unit 20 to irradiate a sample with sheet light traveling in adirection parallel to a surface observed with the observation lens 31.Further, the observation lens 31 has an optical axis in a direction(that is, the Z′ direction in FIG. 1) inclined with respect to thedirection perpendicular to the placement surface 11 (that is, the Zdirection in FIG. 1). FIG. 1 illustrates an embodiment in which the Zdirection and the Z′ direction are inclined with respect to each otherat 15 degrees, and on the X-Z plane, the illumination lens 26 has anoptical axis in a direction inclined at ϕ=15 degrees with respect to theplacement surface 11 (that is, the X-Y plane in FIG. 1). This means thatthe optical axis of the illumination lens 26 is perpendicular to theY-Z′ plane in FIG. 1.

The angle ϕ between the Z direction and the Z′ direction is not limitedto 15 degrees. In a case where the angle between the Z direction and theZ′ direction is larger than 15 degrees, increasing the numericalaperture of the illumination lens 26 allows generation of thinner sheetlight. Such sheet light will, however, have a smaller light collectiondepth. In a case where the angle is smaller than 15 degrees, it ispossible to reduce the aberration discussed above and effectivelyutilize the working distance of the objective lens to observe a deeperportion of a sample. In this case, however, only an objective lens witha smaller numerical aperture is usable, so that while sheet light willhave a larger light collection depth, the sheet light will have a largerthickness. It is thus preferable to adjust the angle between the opticalaxis of the illumination lens 26 and the placement surface 11 within therange from 1 degree to 75 degrees in correspondence with the purpose ofmeasurement. In particular, in a case of observing an object at a lowmagnification (up to ×20), the angle is preferably 1 degree to 10degrees, in a case of observing object at a high magnification (×40 orhigher) over the entire objective lens, the angle is preferably 5degrees to 20 degrees, and in a case of observing an object at a highmagnification (×40 or higher) with a low background, the angle ispreferably 35 degrees to 50 degrees.

The microscope 1 of the present embodiment is arranged such that thesheet light has an optical axis inclined with respect to the placementsurface 11. This arrangement makes it possible to observe a sample 12without influencing any other sample 12 on the sample placement part 10by irradiation with sheet light. The microscope 1 is thus suitable alsofor measurement that uses a multiwell dish or a microfluidic device.

FIG. 2 schematically illustrates cross sections of the sample 12 and thesample placement part 10 on the Y-Z′ plane, which includes the opticalaxis of the observation lens 31, and a region illuminated with sheetlight. FIG. 2 shows a Y direction and a Z′ direction identical to thoseshown in FIG. 1.

The illumination lens 26, as described above, emits sheet light. Thissheet light forms a thin illumination region on its optical path. Asillustrated in FIG. 2, assuming that on the plane perpendicular to theoptical axis of the illumination lens 26 (that is, the Y-Z′ plane inFIG. 2), the illuminated region has a thickness D and a width W, theilluminated region has, on the Y-Z′ plane, a thickness directionidentical to the Z′ direction in FIG. 2. This means that the observationlens 31 is so oriented as to have an optical axis parallel to thethickness direction of the illuminated region (that is, the Z′ directionin FIG. 2). Irradiating a sample 12 with sheet light excites fluorescentmaterial present in the illuminated region of the sample 12, so that thefluorescent material radiates fluorescence. The position of theobservation lens 31 (and that of the illumination lens 26, if necessary)is adjusted so that the focus surface (observation plane) of theobservation lens 31 is included in the illuminated region, and theobservation lens 31 receives fluorescence radiated from the sample 12.

The microscope 1 of the present embodiment allows observation of asample 12 placed on the sample placement part 10 without the need toembed the sample in a gel and in a state where the following advantagesof a SPIM are utilized: (1) A SPIM, which allows observation with weaklight irradiation, allows reduction of harmful action to a sample orattenuation of fluorescence; (2) a SPIM is capable of producing athree-dimensional image rapidly; and (3) a SPIM is capable of observinga sample at a high resolution. Further, the microscope 1 of the presentembodiment is arranged such that the observation lens 31 is so disposedas to be opposite to a sample 12 with respect to the plane including theplacement surface 11. Thus, the area above the sample 12 is open, sothat the size of a sample 12 placed on the sample placement part 10 isnot limited to a size within the range of the working distance of theobservation lens 31. Further, the illumination lens 26 is, as describedabove, so disposed as to be opposite to a sample 12 with respect to theplane including the placement surface 11, so its sheet light irradiatesthe sample 12 through the bottom face 13. This arrangement allows allsheet light to enter the observation plane from the bottom face 13 ofthe sample placement part 10, and thus allows the thickness of sheetlight to be controlled successfully even in a case where theillumination lens 26 has a large numerical aperture.

FIG. 3 is a diagram schematically illustrating an overall configurationand an optical path of the microscope 1 of the present embodiment. Themicroscope 1, as illustrated in FIG. 3, includes the optical unit 20 andan observation section 30 including the observation lens 31. The opticalunit 20 generates sheet light, which irradiates a sample 12. Theobservation section 30 then receives fluorescence from the sample 12 atthe observation lens 31 to form an image. The description below dealswith arrangements of an optical unit and an observation section that arepreferable for use in the overall configuration of the microscope 1 ofthe present embodiment and properties of sheet light for the case inwhich the microscope 1 includes such preferable optical unit andobservation section.

<Optical Unit>

The optical unit 20 includes a visible light source section 21, avisible light adjusting section 22, an IR light source section 23, an IRlight adjusting section 24, a dichroic mirror (DM) 25, and anillumination lens 26 (illumination objective lens).

The visible light source section 21 includes a 592-nm laser light source211, a 560-nm laser light source 212, a 514-nm laser light source 213, a450-nm laser light source 214, a 405-nm laser light source 215, mirrors216 to 219, and DMs 1A to 4A. The visible light source section 21 isarranged such that the laser light sources 211 to 215 emit laser beamshaving different wavelengths, and the laser beams are reflected by thereflecting surfaces of the mirrors 216 to 219 and/or the reflectingsurfaces of the DMs 1A to 4A for reflecting light beams having thewavelengths, so that the optical paths of the laser beams havingdifferent wavelengths are aligned with one another. The visible lightsource section 21 thus emits a composed laser beam to the visible lightadjusting section 22 via a shutter 220 and a mirror 221.

The visible light adjusting section 22 includes a filter foil 222, aone-dimensional scanning mirror 223, a lens 224, an iris 225, a mirror226, and a lens 227. The visible light adjusting section 22 allows alaser beam from the visible light source section 21 to strike theone-dimensional scanning mirror 223 through the filter foil 222. Theone-dimensional scanning mirror 223 is a mirror for scanning the laserbeam one-dimensionally over its reflecting surface. The one-dimensionalscanning mirror 223 is, for example, a MEMS mirror, a piezo mirror, or agalvano mirror. The reflecting surface of the one-dimensional scanningmirror 223 changes one-dimensionally on the basis of a sine function, sothat the light reflected by the one-dimensional scanning mirror 223 is alight beam having an optical path with a fixed width. The visible lightadjusting section 22 is arranged such that light beams are reflected bythe one-dimensional scanning mirror 223 at its reflecting surface,focused by the lens 224, passed through the iris 225, reflected by themirror 226 at its reflecting surface, converted by the lens 227 intoparallel light having a width, and then emitted toward the DM 25. In acase where the visible light source section 21 includes a large numberof laser light sources, an achromat lens can preferably be used for eachof the lens 224 and the lens 227. Further, the one-dimensional scanningmirror 223 may be replaced with, for example, a cylindrical lens, anacousto-optic deflector, or a diffraction grating for adjustment of thewidth of sheet light. In other words, the optical unit 20 for thepresent invention includes a one-dimensional scanning mirror, acylindrical lens, an acousto-optic deflector, a diffraction grating, orthe like as a light distributing element.

The IR light source section 23 includes an IR laser light source section231 and a mirror 232. The IR light source section 23 is arranged suchthat the IR laser light source section 231 emits an IR laser beam, andthe mirror 232 reflects the IR laser beam at its reflecting surface, sothat the IR laser beam is emitted toward the IR light adjusting section24.

The IR light adjusting section 24 includes a one-dimensional scanningmirror 241, an IR lens 242, a mirror 243, an IR lens 244, and a mirror245. The IR light adjusting section 24 is arranged such that the IRlight source section 23 emits an IR laser beam, and the one-dimensionalscanning mirror 241 reflects the IR laser beam at its reflectingsurface, so that the IR laser beam will have an optical path with afixed width. The IR light adjusting section 24 is further arranged suchthat the IR light beam as reflected by the one-dimensional scanningmirror 241 at its reflecting surface is focused by the IR lens 242,reflected by the mirror 243 at its reflecting surface, converted by thelens 244 into parallel light having a width, reflected by the mirror 245at its reflecting surface, and then emitted toward the DM 25. Themicroscope 1 of the present embodiment may alternatively be arrangedsuch that a sample radiates fluorescence through multiphoton excitation.In this case, an achromat lens can preferably be used for each of the IRlens 242 and the IR lens 244 as well. Since such multiphoton excitationrequires a light source for an IR wave range and correction of chromaticaberration, the IR light source section 23 and the IR light adjustingsection 24 each need to be designed independently of the visible lightsource section 21 and the visible light adjusting section 22.

The DM 25 is a dichroic mirror that reflects a visible light beam andpasses an IR light beam through itself. The DM 25 passes, through onesurface thereof, an IR light beam from the IR light adjusting section 24and at the other surface, reflects a visible light beam from the visiblelight adjusting section 22 to align the optical paths of the IR lightbeam and the visible light beam with each other and then emit the IRlight beam and the visible light beam toward the illumination lens 26.

The illumination lens 26 focuses the IR light beam and the visible lightbeam into sheet light and emits such sheet light toward a sample 12.

The individual optical elements included in the optical unit 20 such aslight sources, lenses, and mirrors may be disposed on a surface of anoptical surface plate. In this case, the optical surface plate is sooriented that the surface on which the optical elements are disposed isparallel to the observation plane. This arrangement allows generation ofsheet light traveling in the direction parallel to the observation planeand simplifies the step of adjusting the configuration of the opticalelements. The optical unit 20 of the present embodiment may be attachedto a general upright microscope or inverted microscope for practice ofthe present invention.

The present embodiment has an example configuration in which anillumination lens 26 is disposed at the opening for emission of lightfrom the optical unit 20. The optical unit 20 may, however, beconfigured without the illumination lens 26. In the case where theillumination lens 26 is not used, adjusting the configuration of thelens 227 and the IR lens 244 in FIG. 3 can appropriately change thethickness of sheet light to be emitted by the optical unit 20. Thelenses included in the optical unit 20 may each be replaced with aconical prism or diffraction grating as an optical element (lightconverging element) that focuses light similarly to a lens forgeneration of the sheet light.

<Observation Section>

The observation section 30 includes the observation lens 31 (detectionobjective lens), a fluorescence mirror cube 32, a mirror 33, an imaginglens 34, and a CCD camera 35. The observation lens 31 refracts lightradiated from a sample 12 and emits the refracted light toward thefluorescence mirror cube (fluorescence filter) 32. The fluorescencemirror cube 32 passes, through itself, only the fluorescence from thesample 12 out of the light from the observation lens 31. The observationsection 30 is arranged such that the imaging lens 34 collectsfluorescence from the fluorescence mirror cube 32, and then the mirror33 reflects the collected fluorescence at its reflecting surface, sothat the reflected fluorescence enters the CCD camera 35. Theobservation section 30, through this operation, forms an image fromfluorescence radiated from the sample 12.

<Properties of Sheet Light>

The description below deals with sheet light of the microscope 1 of thepresent embodiment. Fluorescence radiated from that portion of a samplewhich is not the observation plane composes the background for theobservation plane of the observation lens 31. To reduce the background,the illuminated region preferably has a small thickness. The microscope1 of the present embodiment forms an illuminated region having athickness D of 3 μm or less.

FIG. 4 illustrates shapes of sheet light of the microscope 1 of thepresent embodiment. (a) of FIG. 4 shows a diagram (A1) schematicallyillustrating an optical path of sheet light and a fluorescence image(A2) formed by the observation section 30 for a case in which scanningby the one-dimensional scanning mirror is off. (b) of FIG. 4 shows adiagram (B1) schematically illustrating an optical path of sheet lightand a fluorescence image (B2) formed by the observation section 30 for acase in which scanning by the one-dimensional scanning mirror is on. TheX′ direction in FIG. 4 indicates the direction of the optical axis ofthe illumination lens, and the Y direction in FIG. 4 is identical tothat of FIGS. 1 through 3. The fluorescence images of FIG. 4 arefluorescence images that have been produced by irradiating, with laserlight (592 nm), an aqueous solution containing fluorescence molecules(rhodamine) which aqueous solution is sandwiched between the sampleplacement part 10 and a slide glass and with use of the CCD camera 35,capturing an image of fluorescence radiated from the sample. Also byadjusting the scan width of the one-dimensional scanning mirror 233, itis possible to control the width W of a region illuminated with sheetlight. FIG. 4 illustrates a fluorescence image for a case in which theone-dimensional scanning mirror 233 has scanned light beamsone-dimensionally so that the region illuminated with sheet light has awidth W of 100 μm along the Y direction. Further, also by controllingthe iris 225 to open or close it, the width W of the region illuminatedwith sheet light can be controlled. In addition, the illumination lensmay be provided with an aperture stop attached thereto so thatcontrolling the aperture stop can adjust the thickness D and lightcollection depth of the region illuminated with sheet light. Reducingthe opening of the aperture stop increases the thickness D and lightcollection depth of sheet light.

FIG. 5 illustrates properties of sheet light of the microscope 1 of thepresent embodiment. The X′ direction in FIG. 5 is identical to that ofFIG. 4, and the Z′ direction in FIG. 5 is identical to those of FIGS. 1through 3. (a) of FIG. 5 shows images of a sample on the X′-Y plane thatare produced by scanning the sample placement part with respect to sheetlight. (b) of FIG. 5 shows images of a sample on the X′-Z′ plane thatare produced as above. The images of (a) and (b) of FIG. 5 arefluorescence images produced by scanning the sample placement part 10along the Z′ direction in steps of 1 μm for minute fluorescence beads(D=0.04 μm) fixed in 1 weight % of agarose gel and with use of the CCDcamera 35, capturing an image of fluorescence radiated from the sample.

(c) of FIG. 5 is a graph indicative of a distribution of fluorescenceintensity of the images along the X′ direction. (d) of FIG. 5 is a graphindicative of a distribution of fluorescence intensity of the imagesalong the Z′ direction. An analysis of the fluorescence intensitydistributions in (c) and (d) of FIG. 5 shows that the fluorescenceintensity along the X direction has a half-value width of 0.87 μm andthat the fluorescence intensity along the Z direction has a half-valuewidth of 2.6 μm. The results of the analysis prove that the regionilluminated with sheet light by the microscope 1 has a width of 2.6 μmalong the Z direction.

Comparative Examples

FIG. 6 shows diagrams each schematically illustrating a configuration ofa microscope as a comparative example. (a) of FIG. 6 illustrates aconfiguration of a microscope as a first comparative example. (b) ofFIG. 6 illustrates a configuration of a microscope as a secondcomparative example. (c) of FIG. 6 illustrates a configuration of amicroscope as a third comparative example.

The microscope illustrated in (a) of FIG. 6 as the first comparativeexample is arranged such that the observation lens 31′ has an opticalaxis that is not orthogonal to the optical axis of the illumination lens26′ and that is not parallel to the thickness direction of anilluminated region in a plane perpendicular to the optical axis of theillumination lens 26′. This arrangement results in an image beingununiformly out of focus to varying degrees over the observation plane,the image thus having decreased quality evenness. Further, the abovearrangement lets sheet light irradiate that portion of a sample which isnot the observation plane, with the result of a large background.

The microscope illustrated in (b) of FIG. 6 as the second comparativeexample is arranged such that the observation lens 31′ has an opticalaxis that is orthogonal to the optical axis of the illumination lens 26′and that is parallel to the thickness direction of an illuminated regionin a plane perpendicular to the optical axis of the illumination lens26′. The illumination lens 26′ is, however, disposed on the same side asa sample with respect to a plane including the placement surface.

The microscope illustrated in (c) of FIG. 6 as the third comparativeexample is arranged such that the observation lens 31′ has an opticalaxis that is orthogonal to the optical axis of the illumination lens 26′and that is parallel to the thickness direction of an illuminated regionin a plane perpendicular to the optical axis of the illumination lens26′. The illumination lens 26′ and the observation lens 31′ are,however, disposed on the same side as a sample with respect to a planeincluding the placement surface.

The microscope as the second comparative example and the microscope asthe third comparative example are both arranged such that theillumination lens is disposed on the same side as a sample with respectto the plane including the placement surface. Since increasing thenumerical aperture of the illumination lens widens the optical path ofsheet light along the Z direction, the microscope as the secondcomparative example and the microscope as the third comparative examplewill both let part of sheet light be separated by the sample placementpart before illuminating an observation plane. The resulting sheet lightwill have a large thickness and thus illuminate a region of a samplewhich region is other than the observation plane, with the result of alarge background. In particular, in a case of observing a sample in thevicinity of the placement surface, the illumination lens needs to havean optical axis close to the plane including the placement surface sothat the sample will have an illuminated region close to the placementsurface. In a case where the illumination lens has an optical axis closeto the placement surface, sheet light is more likely separated by theplacement surface before illuminating the observation plane. Themicroscopes as the second comparative example and the third comparativeexample are, as described above, incapable of observing a sample at ahigh resolution by increasing the numerical aperture of the illuminationlens. In particular, the microscopes as the second comparative exampleand the third comparative example fail to allow observation of a sampleat a high resolution in the vicinity of the placement surface.

Further Embodiment

The description below deals with another embodiment of the presentinvention with reference to FIGS. 7 through 10. For convenience ofexplanation, any member of the present embodiment that is identical infunction to a corresponding member described for Embodiment 1 isassigned an identical reference numeral, and is not described here.

<Focusing Mechanism>

FIG. 7 is a diagram schematically illustrating a configuration of amicroscope 100 of the present embodiment. The microscope 100, asillustrated in FIG. 7, includes a vibration removal table 101, amicroscope body 102 on the vibration removal table 101, and a condenser103 for irradiating a sample 12 from above.

The microscope 100 further includes a first stage 110 and a second stage120 together as a focusing mechanism for adjusting the relativepositional relationship between a sample placement part 10 and anobservation lens 31. The first stage 110 is placed on the second stage120. The first stage 110 includes a jig for fixing the sample placementpart 10, and the sample placement part 10 is fixed to the first stage110 by means of the jig.

The first stage 110 is capable of changing the position of the sampleplacement part 10 relative to the observation lens 31 along a firstdirection (that is, the Z′ direction in FIG. 7), which is identical tothe direction of the optical axis of the observation lens 31. The secondstage 120 is capable of changing the position of the sample placementpart 10 relative to the observation lens 31 along a second direction andthird direction (that is, the X direction and Y direction in FIG. 7)that define a placement surface of the sample placement part 10.

The microscope 100, as described above, allows adjustment of therelative positional relationship between the observation lens 31 and thesample placement part 10 along each of the first to third directions.The relative positional relationship between the observation lens 31 andthe sample placement part 10 may alternatively be adjusted by fixing theposition of the sample placement part 10, placing the observation lens31 and the illumination lens 26 respectively on the first stage 110 andthe second stage 120, and changing the positions of the observation lens31 and the illumination lens 26.

FIG. 7 illustrates an embodiment in which the first stage with a jigattached thereto is placed on the second stage. The first stage and thesecond stage can function as a focusing mechanism suitable for thepresent invention also in a case where a jig is attached to the secondstage as long as that second stage is placed on the first stage.

FIG. 8 shows diagrams each illustrating how the position of an imagechanges within an identical field of view in response to a change in thepositional relationship between an observation lens and a sampleplacement part. (a) of FIG. 8 illustrates how the position of an imagechanges within an identical field of view in response to a change in theposition of a sample placement part relative to an observation lensalong a direction perpendicular to a placement surface (that is, the Zdirection in FIG. 7). (b) of FIG. 8 illustrates how the position of animage changes within an identical field of view for the microscope ofthe present embodiment in response to a change in the position of thesample placement part relative to the observation lens along thedirection of the optical axis of the observation lens (that is, the Z′direction in FIG. 7).

In a case where the position of a sample placement part is changed alongthe direction perpendicular to a placement surface as illustrated in (a)of FIG. 8, the position of an image undesirably changes within a fieldof view of an observation lens. In contrast, the microscope 100, whichhas the focusing mechanism described above, is arranged such thatchanging the position of the sample placement part along the firstdirection (that is, the direction of the optical axis of the observationlens 31) as illustrated in (b) of FIG. 8 can achieve focus withoutchanging the position of an image within a field of view observed. Thisarrangement makes it possible to continuously capture an image of asample for formation of a three-dimensional image with the image fixedwithin an identical field of view.

The microscope 100, which has the focusing mechanism described above, isarranged such that adjusting the relative positional relationshipbetween the observation lens 31 and the sample placement part 10 alongthe second direction and third direction can change an observed field ofview without changing the distance between the sample placement part 10and the observation lens 31. This arrangement can prevent unnecessarycontact between the sample placement part 10 and the observation lens 31while the microscope 100 changes the observed field of view.

The focusing mechanism as part of the microscope 100 may alternativelybe a separate focusing device for use in a conventionally publicly knownmicroscope.

<Fluid Holding Device>

FIG. 9 shows diagrams each schematically illustrating a mainconfiguration of the microscope 100. (a) of FIG. 9 is a diagramschematically illustrating a sample placement part and an observationlens with a fluid holding device connected thereto. (b) of FIG. 9 is adiagram schematically illustrating a sample placement part and anobservation lens for a case where the sample placement part is separatedfrom the observation lens. (c) of FIG. 9 is a diagram schematicallyillustrating a sample placement part and an observation lens for a casewhere a sample is close to the observation lens.

The microscope 100, as illustrated in (a) of FIG. 9, includes a fluidholding device 40 between the observation lens 31 and the sampleplacement part 10. FIG. 10 shows diagrams each schematicallyillustrating the fluid holding device. (a) of FIG. 10 is a perspectiveview of the fluid holding device. (b) of FIG. 10 is a top view of thefluid holding device as connected to an observation lens, the view beingtaken along the optical axis of the observation lens. (c) of FIG. 10 isa cross-sectional view of the fluid holding device as connected to themicroscope 100 via an observation lens, the view being taken on a planedefined by the optical axis of an illumination lens and the optical axisof the observation lens.

The shape of the fluid holding device 40 is, as illustrated in FIG. 10,a substantially cylindrical shape to fit with the observation lens 31 tobe connected, and its inside has a hollowed structure. The fluid holdingdevice 40 includes a connection section 41 for connection to theobservation lens 31, an end face 42 at such a position as to face thebottom face 13 of the sample placement part 10 so that the sampleplacement part 10 is placed on the end face 42, and a transmissionwindow 43 that allows light to pass therethrough. In a state where thefluid holding device 40 is connected to the observation lens 31, thehollow forms a fluid holding section 44 for holding fluid inside itself.

FIG. 9 illustrates an embodiment in which the fluid holding section 44is filled with water 45, and the observation lens 31 is awater-immersion lens. The fluid holding section 44 may be filled with afluid corresponding to the lens to be used; for example, in a case wherethe observation lens 31 is an oil-immersion lens, the fluid holdingsection 44 may be filled with an immersion oil. In a case where theobservation lens 31 is a dry lens, the fluid holding device 40 may beused, or need not be used.

When the microscope 100 is in a state where the fluid holding device 40and the sample placement part 10 are so positioned that the end face 42faces the bottom face 13, the fluid holding device 40 may hold fluid inthe fluid holding section 44 together with the bottom face 13. In thisstate, the end face 42 is substantially parallel to the bottom face 13of the sample placement part 10. A portion of the water 45 is heldbetween the end face 42 and the bottom face 13 as illustrated in FIG. 9due to the surface tension of the water 45 filling the fluid holdingsection 44.

In a case where the microscope 100 is in a state where the fluid holdingdevice 40 is connected to the observation lens 31, the illumination lens26 disposed outside the fluid holding device 40 may irradiate a sample12 with sheet light through the transmission window 43 and the fluidholding section 44. Specifically, the fluid holding device 40 includes afluid holding section 44 that is a hollow extending from thetransmission window 43 to the end face 42 such that sheet light from theillumination lens 26 which sheet light has passed through thetransmission window 43 irradiates the sample 12. Further, the fluidholding device 40 includes a fluid holding section 44 that is a hollowextending, in the state where the fluid holding device 40 is connectedto the observation lens 31, from the end face 42 to the connectionsection 41 such that fluorescence from the sample 12 is received by theobservation lens 31. As described above, the microscope 100, whichincludes the fluid holding device described above, is capable of holdingfluid such as liquid or gas between the observation lens 31 and thesample placement part 10 while ensuring a path of sheet light and a pathof fluorescence. Further, by filling the fluid holding section 44 withliquid, the shape of the surface of the liquid does not change when, forexample, the sample 12 is moved, and the surface of the liquid has astable shape in correspondence with the shape of the fluid holdingsection 44. As described above, in the case where the microscope 100includes an immersion lens as the observation lens 31, the microscope100, which includes the fluid holding device described above, canirradiate a sample 12 with sheet light through a liquid having a stablesurface shape, and can thus form an illuminated region at a desiredposition.

The fluid holding device as part of the microscope 100 may alternativelybe a separate fluid holding device for use in a conventionally publiclyknown microscope.

<Sample Placement Part>

FIG. 11 shows diagrams each schematically illustrating a mainconfiguration of the microscope 100. The diagrams each illustrate anoptical path of fluorescence from a sample, the angle between a sampleplacement part and the optical axis of an observation lens, and an imageformed by an observation section.

In a case where the observation lens is an immersion lens, the spacebetween the sample placement part and the observation lens is filledwith a desired liquid. In this case, the difference between therefractive index of the sample placement part and that of theabove-mentioned liquid causes the optical path of fluorescence from asample to change by refraction. Thus, even though the sample placementpart has an observation plane perpendicular to the optical axis of theobservation lens, aberration will emerge depending on the numericalaperture of the observation lens, with the result of unstable imaging(see (a) of FIG. 11). Such aberration may be removed with use of acorrection collar for the observation lens. However, in a case where thesample placement part has an observation plane not perpendicular to theoptical axis of the observation lens (that is, the sample placement partis inclined from a plane perpendicular to the optical axis of theobservation lens), left-right asymmetric aberration will emerge which isextremely difficult to remove (see (b) of FIG. 11).

The microscope 100 is, in the case where the observation lens is awater-immersion lens, preferably arranged such that the sample placementpart is made of a material having a refractive index equivalent to thatof water (1.28 to 1.38) which material is typified by an amorphousfluorine resin having the following structural formula:

The above arrangement can prevent light refraction at the sampleplacement part and can thus significantly reduce aberration that occursdepending on the numerical aperture of the observation lens, therebyallowing the microscope to form a stable image (see (c) of FIG. 11).Further, even though the sample placement part has an observation planenot perpendicular to the optical axis of the observation lens, the abovearrangement can prevent the emergence of left-right asymmetricaberration (see (d) of FIG. 11). The above resin is commerciallyavailable as CYTOP (registered trademark). The sample placement part ofthe microscope 100 may alternatively be made of another fluorine-basedtransparent resin. Further, Lumox may alternatively be used as thesample placement part. In this case, even though the sample placementpart is to be made of a material having a refractive index differentfrom that of water, reducing the thickness of the sample placement partcan reduce effect of refraction.

Specific preferable examples of the sample placement part are describedabove. The sample placement part is, however, not limited to those. Thesample placement part may suitably be any planar substrate that is thinand transparent and that has a refractive index (1.28 to 1.38)equivalent to that of water. The sample placement part 10, which is inthe shape of a planar substrate, requires both strength to hold a sampleand thinness sufficient to prevent influence on observation. Thethickness is preferably within the range from 1 μm to 200 μm. Inparticular, in a case of observing a sample on a sample placement part10 as Lumox (registered trademark, with a refractive index of 1.36), thesample placement part 10 preferably has a thickness within the rangefrom 10 μm to 50 μm to reduce aberration caused by the sample placementpart 10 and to allow stable observation. In a case of observe a sampleon a sample placement part 10 made of CYTOP (registered trademark, witha refractive index of 1.34), the sample placement part 10 preferably hasa thickness within the range from 10 μm to 100 μm to reduce aberrationcaused by the sample placement part 10 and to allow stable observation.

<Further Arrangement>

The description below deals with other features of the microscopes 1 and100 of the present invention.

(Visible Light Adjusting Section)

The microscopes 1 and 100 of the present invention are both arrangedsuch that the one-dimensional scanning mirror 233 reflects, at itsreflecting surface, light from the laser light sources 211 to 215 touniformly distribute light in the Y-axis direction shown in FIGS. 12 and13.

FIG. 12 shows diagrams each illustrating an optical path and a luminancedistribution of a light beam in the optical unit. (a) of FIG. 12illustrates a light beam reflected at the reflecting surface of aone-dimensional scanning mirror and a light luminance distribution. (b)of FIG. 12 illustrates a light beam reflected at the reflecting surfaceof a one-dimensional scanning mirror and passing through an iris and alight luminance distribution. The one-dimensional scanning mirror 233scans its reflecting surface one-dimensionally on the basis of a sinefunction. Thus, as illustrated in (a) of FIG. 12, light reflected by theone-dimensional scanning mirror 233 has higher luminance at edges in thewidth direction than in the middle of the light beam. The microscopes 1and 100 of the present embodiment are, in view of that, both arranged toproduce light with uniform luminance as illustrated in (b) of FIG. 12 byreflecting light at the one-dimensional scanning mirror 233, collectingthe light at the lens 224, and then passing the light through the iris225 to remove edges of the light beam in the width direction.

<Variations>

The description below deals with variations of the present inventionwith reference to FIGS. 13 through 15. For convenience of explanation,any member of the present embodiment that is identical in function to acorresponding member described for Embodiment 1 is assigned an identicalreference numeral, and is not described here.

FIG. 13 is a diagram schematically illustrating an overall configurationand an optical path of a microscope 200 of a variation of the presentinvention. The microscope 200, as illustrated in FIG. 13, includes asample placement part 10, an optical unit 20A, and an observationsection 30A.

The optical unit 20A includes a visible light source section 21A, avisible light adjusting section 22A, an IR light source section 23A, anIR light adjusting section 24A, a dichroic mirror (DM) 25, anillumination lens 26 (illumination objective lens), and a wedge prism50.

The visible light source section 21A is arranged such that the laserlight sources 211 to 215 emit laser beams having different wavelengthsand that the laser beams are reflected by the reflecting surface of amirror 216 and/or the reflecting surfaces of DMs 1A to 4A for reflectinglight beams having the wavelengths, so that the optical paths of thelaser beams having different wavelengths are aligned with one another.The visible light source section 21A is arranged such that the laserbeams having the wavelengths and optical paths aligned with one anotherare reflected at the reflecting surface of a mirror 217 and are thencomposed with use of fiber coupler 60 s and a fiber 61. The visiblelight source section 21A thus emits a composed laser beam to the visiblelight adjusting section 22A via a shutter 220.

The visible light adjusting section 22A includes mirrors 70 to 75, amirror 221, DMs 1B to 4B, DMs 1C to 4C, a filter foil 222, aone-dimensional scanning mirror 223, a lens 224, an iris 225, a mirror226, and a lens 227. The visible light adjusting section 22A is arrangedto reflect laser beams from the visible light source section 21A at thereflecting surface of the mirror 70 and then reflect the laser beams atthe reflecting surfaces of the DMs 1B to 4B, mirrors 71 to 75, and DMs1C to 4C at different reflection angles corresponding to the wavelengthsso as to separate, from one another, the optical paths of the laserbeams having the different wavelengths. The visible light adjustingsection 22A allows the laser beams to strike the one-dimensionalscanning mirror 223 through the filter foil 222, and thus allowsparallel light having a fixed width to be emitted toward the DM 25.

The IR light source section 23A and IR light adjusting section 24A arethe same respectively as the IR light source section 23 and IR lightadjusting section 24 of Embodiment 1. The IR light adjusting section 24Aemits an IR light beam toward the DM 25.

The DM 25 passes an IR light beam from the IR light adjusting section24A through one surface thereof and at the other surface, reflects avisible light beam from the visible light adjusting section 22A to alignthe optical paths of the IR light beam and the visible light beam witheach other and to then emit the IR light beam and the visible light beamtoward the wedge prism 50. The wedge prism 50 bends the IR light beamand the visible light beam and then emits the IR light beam and thevisible light beam toward the illumination lens 26.

The microscope 1 of Embodiment 1 is, in order to irradiate a sample 12with sheet light through the bottom face 13 of the sample placement part10, arranged such that as illustrated in FIG. 3, the illumination lens26 has an optical axis inclined at the angle ϕ with respect to theplacement surface 11, and the other members of the optical unit 20 arealso disposed at the angle ϕ. Assuming that the Z direction in FIG. 3 isthe vertical direction, it will not be easy to dispose the individualmembers of the optical unit 20 with reference to the Z′ direction, whichis inclined at the angle ϕ from the Z direction.

In view of that, as the microscope 200 of the variation, the microscope1 of Embodiment 1 may be equipped with a wedge prism 50 provided betweenthe DM 25 and the illumination lens 26 to bend light beams between theDM 25 and the illumination lens 26. This arrangement eliminates the needto adjust the orientations of the individual members of the optical unit20 in correspondence with the angle ϕ, thereby facilitating assembly ofthe optical unit 20.

FIG. 14 illustrates arrangements each for bending a light beam. (a) ofFIG. 14 illustrates an arrangement for bending a light beam byreflecting the light beam at the reflecting surface of a mirror. (b) ofFIG. 14 illustrates an arrangement for bending a light beam by passingthe light beam through a wedge prism. In a case where a beam of lightfor forming an image of the letter R is bended through reflection at thereflecting surface of a mirror as illustrated in (a) of FIG. 14, theimage is inclined with respect to a plane (irradiation plane)perpendicular to the axis of the light beam. In a case where a lightbeam having a fixed width as in the optical unit 20 of each of themicroscopes 1 and 100 of the present invention is bended throughreflection at the reflecting surface of a mirror, the width direction ofthe light beam is undesirably changed. In this case, it is impossible tosuccessfully control the thickness of a region illuminated with sheetlight emitted by the illumination lens 26.

In contrast, in the microscope 1, a light beam having a fixed widthpasses through the wedge prism 50 to be bended without changes in itswidth direction.

FIG. 15 shows diagrams each illustrating optical paths of light beamshaving different wavelengths which light beams are bended with use of awedge prism in an optical unit. (a) of FIG. 15 illustrates how themutually aligned optical paths of multispectral laser beams are changedwhen the multispectral laser beams pass through a wedge prism. (b) ofFIG. 15 illustrates how the mutually varied optical paths ofmultispectral laser beams are changed when the multispectral laser beamspass through a wedge prism.

The optical unit 20 of the microscope 1 of the variation uses a wedgeprism 50 to bend light beams. In such a case, since the wedge prism 50has a refractive index that varies depending on the wavelength of light,when different laser beams having different wavelengths and having thesame optical path are bended by being passed through the wedge prism 50,the optical paths of the laser beams having passed through the wedgeprism 50 would be different from one another with respect to thewavelengths as illustrated in (a) of FIG. 15. In this case, sheet lightemitted by the optical unit 20 undesirably forms an illuminated regionhaving a large thickness.

The optical unit 20 is thus preferably arranged as illustrated in (b) ofFIG. 15 such that light beams having different wavelengths and havingdifferent optical paths aligned with one another are first bended withuse of DMs, mirrors, and lenses at angles predetermined incorrespondence with respect to each wavelength so that the optical pathsdiffer from one another in correspondence with respect to eachwavelength and that the light beams are then bended with use of a wedgeprism 50. This arrangement involves adjusting the optical paths of lightbeams with respect to each wavelength in advance with use of DMs,mirrors, and lenses so that the light beams having the differentwavelengths which light beams have passed through the wedge prism willhave optical paths aligned with one another.

Example 1

FIG. 16 illustrates an Example involving use of a microscope of thepresent invention. (a) of FIG. 16 shows images of a budding yeast cellon different X′-Y planes which images were produced by scanning anobservation plane along the Z′ direction. (b) of FIG. 16 shows athree-dimensional image of the budding yeast cell.

The images of FIG. 16 are fluorescence images produced by irradiating,with sheet light, a budding yeast cell (Saccharomyces cerevisiae:mKate2/pESC-HIS/BY4741) clustered in the vicinity of the placementsurface of the sample placement part while scanning the illuminatedregion along the first direction (Z′ direction) in steps of 0.25 μm withuse of the first stage 110 and with use of the CCD camera 35, capturingan image of fluorescence radiated from the sample.

The microscope of the present invention allows observation of a samplein the vicinity of the placement surface of the sample placement part(cover slip). The microscope successfully formed an image of the entirevolume (approximately 6 μm in diameter) of a budding yeast cell in thevicinity of the placement surface as described above.

Example 2

FIG. 17 illustrates an Example involving use of a microscope of thepresent invention. (a) of FIG. 17 is a fluorescence image of Escherichiacoli. (b) of FIG. 17 is a luminance profile for the fluorescence imageof Escherichia coli.

The microscope of the present invention can include a lens with a largenumerical aperture (for example, NA=1.1) as the observation lens 31, andthus allows observation at a high resolution.

Fluorescence imaging was carried out of an Escherichia coli strain (E.coli: SX4) that expresses an average of one fluorescent protein moleculeper cell. A fluorescence image was produced in this observation exampleby fixing Escherichia coli to a cover slip having a low refractive indexand irradiating the sample with 514-nm sheet light to excite the sample.This observation resulted in detection of two bright points ofsingle-molecule fluorescence in the cell 1, no such bright point in thecell 2, and one such bright point in the cell 3 (see FIG. 17).

The present invention is not limited to the description of theembodiments above, and may be altered within the scope of the claims invarious ways. Any embodiment based on a proper combination of technicalmeans disclosed in different embodiments is also encompassed in thetechnical scope of the present invention.

INDUSTRIAL APPLICABILITY

The present invention allows observation of a sample at a highresolution at a single-molecule level which observation is free fromrestrictions such as the size of a sample. The present invention istherefore suitably applicable to development of new medicaments based oncells.

REFERENCE SIGNS LIST

-   -   1, 100 microscope    -   10 sample placement part    -   11 placement surface    -   12 sample    -   13 bottom face    -   20 optical unit    -   31 observation lens    -   40 fluid holding device    -   41 connection section    -   42 end face    -   43 transmission window    -   44 fluid holding section    -   110 first stage (focusing mechanism, focusing device)    -   120 second stage (focusing mechanism, focusing device)

The invention claimed is:
 1. A microscope, comprising: a sampleplacement part having a placement surface at which to place a sample anda face opposite to the placement surface; an observation lens forreceiving fluorescence from the sample; a fluid holding device forholding fluid between the observation lens and the sample placementpart; and an optical unit provided outside of the observation lens andconfigured to generate sheet light such that the sheet light enteringthe sample placement part from the face opposite to the placementsurface from an oblique direction with respect to the placement surfacepasses through the sample placement part, and the passed sheet lightirradiates the sample, and the fluorescence passes through the sampleplacement part toward the face opposite to the placement surface, andthe passed fluorescence is received by the observation lens.
 2. Themicroscope according to claim 1, further comprising: a focusingmechanism for adjusting a relative positional relationship between theobservation lens and the sample, wherein the focusing mechanism isconfigured to change the positional relationship along a firstdirection, which is substantially parallel to an optical axis of theobservation lens, and a second direction and a third direction, whichdefine the placement surface.
 3. The microscope according to claim 1,wherein the observation lens is an immersion lens.
 4. The microscopeaccording to claim 3, wherein the fluid holding device is filled with aliquid corresponding to the immersion lens.
 5. The microscope accordingto claim 4, wherein: the fluid holding device is filled with water; andthe sample placement part has a refractive index of 1.28 to 1.38.
 6. Themicroscope according to claim 1, wherein an observation plane and theplacement surface form an angle within a range from 1 degree to 75degrees.
 7. The microscope according to claim 1, wherein: the opticalunit comprises an optical surface plate having a surface on which one ormore optical elements for generating the sheet light are disposed; andthe optical surface plate is oriented so that the surface of the opticalsurface plate is substantially parallel to an observation plane.
 8. Themicroscope according to claim 7, wherein: the one or more opticalelements comprise a light source, a light converging element, and alight distributing element; and the one or more optical elementsoptionally further comprise a wedge prism.